Proxisome proliferator activated receptor (PPAR) compounds and methods of using the same

ABSTRACT

Peroxisome proliferator activated receptor (PPAR) compounds, and methods of using the same for treating bone fractures, treating osteoporosis and/or metabolic bone diseases, and inducing osteogenesis and/or chondrogenesis, are disclosed.

RELATED APPLICATIONS

The present application is divisional application of U.S. Ser. No.14/733,022 filed Sep. 4, 2015, now allowed, which is a national stageapplication filed under 35 U.S.C. § 371 of international applicationPCT/US2014/027817, filed under the authority of the Patent CorporationTreaty on Mar. 14, 2014, which claims priority to U.S. ProvisionalApplication Ser. No. 61/786,030, filed under 35 U.S.C. § 111(b) on Mar.14, 2013, the disclosure of which is incorporated herein by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made with no government support. The government has norights in the invention.

TECHNICAL FIELD

The present disclosure relates to the field of compounds, compositions,and methods useful for the treatment or prevention of osteoporosis,osteoarthritis, metabolic bone disorders, fracture management, and othermusculoskeletal disorders.

BACKGROUND OF THE INVENTION

Osteoporosis is a silent disease of bones that affects tens of millionsof people over the age of 50. The disease results in decreased bonemineral density and ultimately bone fracture. Osteoporosis can lead toacute and chronic fractures, causing significant morbidity and mortalityto patients. Other metabolic bone diseases can similarly result inweakened bones and fractures. Currently, the best medications availablecan reduce recurrent fracture risk only 65% of the time, and areassociated with significant risks such as avascular necrosis of thetempormandibular joints, subtrochanteric femur fractures, and malignantbone tumors.

Osteoarthritis is the most common joint disorder in the world, andaffects the majority of people over the age of 65. Osteoarthritis is adisease of cartilage and bone that results in the wearing away of thelining of the joint, and ultimately bone-on-bone changes. Osteoarthritiscan lead to crippling joint pain and deformity, causing significantmorbidity to patients. Currently, there are no medical treatmentsavailable to prevent or halt the progression of osteoarthritis. Thestandard of care for treating osteoarthritis dictates supportive painmanagement measures such as medications, physical therapy, braces,lifestyle changes, and activity modifications, until a patient can nolonger tolerate the pain, at which point a joint fusion or replacementmay be performed.

It would be advantageous to develop effective ways of preventing ortreating osteoporosis, osteoarthritis, metabolic bone disorders,fracture management, and other musculoskeletal disorders.

SUMMARY OF THE INVENTION

Provided herein are analogs of PPARδ and 20-hydroxy prostaglandin E₂(20-OH-PGE₂).

Provided herein is a method of inducing osteogenesis or chondrogenesis,the method comprising treating mammalian stem cells with an effectiveamount of one or more of a PPARδ agonist and a 20-OH-PGE₂ antagonist,whereby the mammalian stem cells differentiate into a cell of osteoblastor chondroblast lineage

In certain embodiments, the PPARδ agonist comprises the structuralformula of Formula I:

wherein X is S, O, or NH; R is OCH₂R³, CH═CHR³, or (CH₂)_(n)R³, where nis 0, 1, 2, or 3 and R³ is carboxylic acid, sulfonic acid, an acidicsulfonamide, or pharmacophoric mimics thereof; R¹ is H, CH₃, or CF₃; andR² is H, alkyl, substituted alkyl, or halogen; and salts, isomers,stereoisomers, enantiomers, racemates, solvates, hydrates, polymorphs,and prodrugs thereof. In particular embodiments, X is NH.

In certain embodiments, the PPARδ agonist has the structural formula ofFormula I-A:

wherein R₁ is hydrogen and R₂ is CF₃.

In certain embodiments, the 20-OH-PGE₂ antagonist comprises thestructural formula of Formula II:

wherein R is carboxylic acid, sulfonic acid, an acidic sulfonamide, orpharmacophoric mimics thereof; R¹ is H, CH₃, OH, CH₂OH; and n is 1, 2,or 3; and salts, isomers, stereoisomers, enantiomers, racemates,solvates, hydrates, polymorphs, and prodrugs thereof.

In certain embodiments, the stem cells are present in a mammaliansubject. In certain embodiments, the stem cells are isolated stem cells,and the method further comprises the step of administering the treatedmammalian stem cells to a mammal in need thereof.

Further provided herein is a compound having the structural formula ofFormula I:

wherein X is S, O, or NH; R is OCH₂R³, CH═CHR³, or (CH₂)_(n)R³, where nis 0, 1, 2, or 3 and R³ is carboxylic acid, sulfonic acid, an acidicsulfonamide, or pharmacophoric mimics thereof; R¹ is H, CH₃, or CF₃; andR² is H, alkyl, substituted alkyl, or halide; provided that when R isOCH₂CO₂H, (i) either R¹ is not para-CF₃ or R² is not H, and (ii) eitherR¹ is not meta-CH₃ or R² is not para-tert-butyl. Further provided aresalts, isomers, stereoisomers, enantiomers, racemates, solvates,hydrates, polymorphs, and prodrugs thereof. In certain embodiments, X isNH.

In certain embodiments, the compound has the structural formula ofFormula I-A:

wherein R₁ is hydrogen, and R₂ is CF₃.

Further provided herein is a compound having the structural formula ofFormula II:

wherein R is carboxylic acid, sulfonic acid, an acidic sulfonamide, orpharmacophoric mimics thereof; R¹ is H, CH₃, OH, CH₂OH; and n is 1, 2,or 3; provided that when R is CO₂H, either R¹ is not CH₂OH or n is not3. Further provided are salts, isomers, stereoisomers, enantiomers,racemates, solvates, hydrates, polymorphs, and prodrugs thereof.

Further provided is a pharmaceutical composition comprising a compounddescribed herein and a pharmaceutically acceptable carrier, excipient,diluent, or adjuvant. In certain embodiments, the compound is present ata concentration ranging from about 0.1 μM to about 1 μM.

Further provided is a method of treating, ameliorating, or preventosteoarthritis, osteoporosis, or metabolic bone disease, the methodcomprising administering an effective amount of the pharmaceuticalcomposition described herein to a patient in need thereof.

Further provided is a method of treating, ameliorating, or preventingosteoarthritis, osteoporosis, or metabolic bone disease, the methodcomprising treating isolated stem cells with the pharmaceuticalcomposition described herein, and administering the treated stem cellsto a patient in need thereof to treat, ameliorate, or preventosteoarthritis, osteoporosis, or metabolic bone disease.

Further provided is a kit for preparing a pharmaceutical compositioncomprising a first container housing one or more of a PPARδ agonist or a20-OH-PGE₂ antagonist; and a second container housing a pharmaceuticallyacceptable carrier, excipient, diluent, or adjuvant. In certainembodiments, the kit further comprises a syringe configured to inject apharmaceutical composition. In certain embodiments, the kit comprisesboth a PPARδ agonist and a 20-OH-PGE₂ antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIGS. 1A-1C: Levels of mRNA for Cyp4F11 (FIG. 1A) and Cyp4F2 (FIG. 1B),and levels of PGE₂ in mesenchymal stem cells (MSC) (FIG. 1C) before andafter adipogenic differentiation (adipocytes). The data are expressed asmeans±SE. *P<0.05 versus MSC; ^(#)P<0.05 versus vehicle; ^(t)P<0.05versus COX-1 or COX-2 inhibitor.

FIGS. 2A-2B: Effect of 20-HETE on adipogenesis in the presence andabsence of indomethacin, COX-1 inhibitor (valeroyl salicylate), andCOX-2 inhibitor (CAY10404). (FIG. 2A.) Adipogenesis was measured as therelative absorbance of Oil Red O at day 14 after inducing adipogenesis.(FIG. 2B.) Mean±SE, *P<0.05 versus vehicle.

FIG. 3: Effect of 20-HETE agonist on adipogenesis in the presence andabsence of COX-1 or COX-2 inhibitor. Adipogenesis was measured as therelative absorbance of Oil Red O at day 14 after inducing adipogenesis.Mean±SE.

FIGS. 4A-4B: Effect of 20-HETE on adipogenesis in the presence andabsence of COX-1 inhibitor, COX-2 inhibitor, and the PGE₂ synthaseinhibitor (CAY10526). (FIG. 4A.) Adipogenesis was measured as therelative absorbance of Oil Red O at day 14 after inducing adipogenesis.(FIG. 4B.) Mean±SE, *P<0.05 versus vehicle, ^(#)P<0.05 versus control.

FIGS. 5A-5B: Effect of 20-OH-PGE₂ on adipogenesis in the presence andabsence of COX-1 inhibitor and/or COX-2 inhibitor. (FIG. 5A.)Adipogenesis was measured as the relative absorbance of Oil Red O at day14 after inducing adipogenesis. (FIG. 5B.) Mean±SE, *P<0.05 versusvehicle, ^(#)P<0.05 versus control.

FIG. 6A: Concentration-dependent effect of 20-OH-PGE₂ on adipogenesis.Mean±SE, *P<0.05 versus 1 nM concentration of 20-OH-PGE₂. Mean±SE,*P<0.05, and **P<0.01 versus control.

FIG. 6B: Effect of 20-OH-PGE₂ on adipocyte size. Mean±SE, *P<0.05, and**P<0.01 versus control.

FIGS. 7A-7D: Effect of 20-OH-PGE₂ on adipogenic markers. (FIG. 7A.)Expression of PPARγ (FIG. 7B), Mest (FIG. 7C), and β-catenin (FIG. 7D)was determined by Western blot analysis in MSC-derived adipocytes.Quantitative densitometry evaluation of the proteins ratio wasdetermined. Data are expressed as means±SE, *P<0.05 versus correspondingconditions without 20-OH-PGE₂.

FIG. 8: Concentration-dependent effect of 20-OH-PGE₂ on adipogenesis andosteogenesis.

FIG. 9: Stained mesenchymal cells exposed to varying concentrations of20-OH-PGE₂ and a PPARγ agonist.

FIG. 10A: Osteogenesis effects of 20-OH-PGE₂ on mesenchymal cellsstained with Alizarin S and Oil Red O.

FIG. 10B: Effect of 20-OH-PGE₂ on osteogenesis from BM-derivedmesenchymal stem cells. *Control versus 1 μM, P<0.01.

FIGS. 11A-11B: hBM-derived MSCs treated with the PPARγ agonist (GW0742)and PPARγ antagonist (GSK0660) every 2 days during osteogenesis.Increased red staining on GW0742 at 0.1 μM and 1 μM shows an increase inosteogenesis. Decreased red staining on GSK0660 at 1 μM shows a decreasein osteogenesis. *p<0.01, †p<0.001.

FIG. 12: Scheme 1, showing a non-limiting example of a synthetic routeto produce compounds 6a-6c, wherein R₁ is H, CF₃, or CH₃; R₂ is H, CF₃,alkyl, or substituted alkyl.

FIG. 13: Scheme 2, showing a non-limiting example of a synthetic routeto produce various PPARγ agonist analogs, wherein R₁ is H, CF₃, or CH₃;R₂ is H, CF₃, alkyl, or substituted alkyl; R₃ is H, CF₃, alkyl, orsubstituted alkyl.

FIG. 14: Scheme 3, showing a short synthesis of 20-OH-PGE₂ analogs,where R is carboxylic acid, sulfonic acid, or acidic sulfonamide, orpharmacophoric mimics thereof; R₁ is H, CH₃, OH, or CH₂OH; n is 1, 2, or3; and R₂ is a suitable hydroxyl protecting group that allows forsimultaneous removal as the final step. An alternative, 17-stepsynthesis of these compounds is also possible.

FIG. 15: Table 1, showing the differential effects on stem cells whentreated with GW0742 and compounds 6a-6c.

FIG. 16: Effects of GW0742, compound 6a, compound 6b, and compound 6c onadipogenesis and osteogenesis.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments are described herein in the context of PPARδ and20-OH-PGE₂ analogues, and methods of using the same. Those of ordinaryskill in the art will realize that the following detailed description ofthe embodiments is illustrative only and not intended to be in any waylimiting. Other embodiments will readily suggest themselves to suchskilled persons having the benefit of this disclosure. Reference to an“embodiment,” “aspect,” or “example” herein indicate that theembodiments of the invention so described may include a particularfeature, structure, or characteristic, but not every embodimentnecessarily includes the particular feature, structure, orcharacteristic. Further, repeated use of the phrase “in one embodiment”does not necessarily refer to the same embodiment, although it may.

In the interest of clarity, not all of the routine features of theimplementations or processes described herein are shown and described.It will, of course, be appreciated that in the development of any suchactual implementation, numerous implementation-specific decisions willbe made in order to achieve the developer's specific goals, such ascompliance with application- and business-related constraints, and thatthese specific goals will vary from one implementation to another andfrom one developer to another. Moreover, it will be appreciated thatsuch a development effort might be complex and time-consuming, but wouldnevertheless be a routine undertaking for those of ordinary skill in theart having the benefit of this disclosure.

Definitions

The term “PPAR” refers to Peroxisome Proliferator Activated Receptors,which are orphan receptors belonging to the steroid/retinoid receptorsuperfamily of ligand-activated transcription factors. Three mammalianPPARs have been identified, termed PPARα, PPARγ, and PPARδ. PPARsregulate expression of target genes by binding to DNA response elementsas heterodimers with the retinoid X receptor.

The term “pharmacophoric mimic” refers to a compound or functional grouphaving the steric and electronic features necessary for molecularrecognition by a biological macromolecule similar to that of anothercompound or functional group.

The term “alkyl” as used herein refers to monovalent alkyl groups, whichare saturated hydrocarbons, preferably having from 1 to 10 carbon atomsand more preferably 1 to 6 carbon atoms. This term is exemplified bygroups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,n-hexyl, and the like.

Unless stereochemistry is specifically indicated, all stereoisomers ofthe compounds herein are included, as pure compounds as well as mixturesthereof.

Some of the crystalline forms for the compounds may exist as polymorphsand as such are included. In addition, some of the compounds herein mayform solvates with water (i.e., hydrates) or common organic solvents,which are also included.

Protected forms of the compounds herein are further included. A varietyof protecting groups are possible.

Prodrugs of the compounds herein are included. In general, such prodrugsare functional derivatives of the compounds that are readily convertiblein vivo into the required compound. Thus, in the methods of treatment,the term “administering” includes the treatment of the various disordersdescribed with the compound specifically disclosed or with a compoundwhich may not be specifically disclosed, but which converts to thespecified compound in vivo after administration to a subject in needthereof. A simple example of a prodrug, not meant to be limiting in anymanner, would be an alkyl ester of the acidic groups container at R³within Formula I or at R within Formula II.

The term “solvate” refers to a pharmaceutically acceptable solid form ofa specified compound containing solvent molecules as part of the crystalstructure. A solvate typically retains at least some of the biologicaleffectiveness of such compound. Solvates can have differentsolubilities, hygroscopicities, stabilities, and other properties.Examples of solvates include, but are not limited to, compounds incombination with water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, or ethanolamine. Solvates are sometimes termed“pseudopolymorphs.” The term “hydrate” refers to a solvate with water.

The term “racemate” refers to a mixture that contains an equal amount ofenantiomers.

It will be appreciated by one of ordinary skill in the art thatasymmetric centers may exist in any of the compounds disclosed herein.Thus, the compounds may be in the form of an individual enantiomer,diastereomer, or geometric isomer, or may be in the form of a mixture ofstereoisomers. In certain embodiments, the compounds are enantiopurecompounds. In certain other embodiments, mixtures of stereoisomers ordiastereomers are provided. Additionally, the compounds encompass both(Z) and (E) double bond isomers (or cis and trans isomers) unlessotherwise specifically designated. Thus, compounds generally depicted instructures herein encompass those structures in which double bonds are(Z) or (E).

It will also be appreciated that any of the compounds described hereinmay be substituted with any number of substituents or functionalmoieties. In general, the term “substituted” whether preceded by theterm “optionally” or not, and substituents contained in formulas, referto the replacement of hydrogen atoms in a given structure with aspecified substituent. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents. For purposes of explanation herein, heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms. Furthermore, there is not any intention tobe limited in any manner by the permissible substituents.

General Description

Mesenchymal stem cells are stem cells that can develop into connectivetissue throughout the body, such as bone, fat, and cartilage. Thepresent disclosure is aimed at directing mesenchymal stem cells towardosteogenesis or chondrogenesis as opposed to adipogenesis, therebyinducing bone formation over fat formation. The compounds, compositions,and methods described herein are thus useful in the treatment and/orprevention of musculoskeletal disorders such as osteoporosis,osteoarthritis, metabolic and bone disease, as well as for fracturemanagement and prosthetic integration.

Further provided herein is a method of inducing osteogenesis, the methodcomprising: contacting a mammalian cell with an effective amount of atleast one pharmaceutical composition described herein, whereby themammalian cell differentiates into a cell of an osteoblast lineage, orwhereby the mammalian cell differentiates into a cell of a chondroblastlineage.

In certain embodiments, the mammalian cell is an in vivo mammalian cell.

In certain embodiments, the mammalian cell is a mesenchymal stem cell.

In certain embodiments, the stem cell is isolated from a primate.

In certain embodiments, the primate is a human.

In certain embodiments, the step of contacting is by oral administrationof the compound to the mammal.

In certain embodiments, the step of contacting is by intravenousadministration of the compound to the mammal.

In certain embodiments the step of contacting is by subcutaneousadministration of the compound to the mammal.

In certain embodiments, the method further comprises detectingdifferentiation of the mammalian cell into an osteocyte cell of anosteoblast lineage.

In certain embodiments, the method further comprises detectingdifferentiation of the mammalian cell into a chondrocyte cell of achondroblast lineage.

In certain embodiments, wherein the mammalian cell is attached to asolid support.

In certain embodiments, the solid support is a three dimensional matrix.

In certain embodiments, the solid support is a planar surface.

Further provided herein is a method of treating a bone disorder,comprising: contacting a mammalian cell with a pharmaceuticalcomposition as described herein, whereby the mammalian celldifferentiates into a cell of an osteoblast lineage, wherein the bonedisorder is associated with defective osteoblasts.

In certain embodiments, the bone disorder is osteoporosis.

In certain embodiments, the method further comprises administering thecell of an osteoblast lineage to an individual with the disorder,thereby treating the disorder.

In certain embodiments, the administration is by surgical implantation.

Further provided herein is a method of treating a cartilage disorder,comprising: contacting a mammalian cell with a pharmaceuticalcomposition as described herein, whereby the mammalian celldifferentiates into a cell of a chondroblast lineage, wherein the bonedisorder is associated with defective chondroblasts.

In certain embodiments, the cartilage disorder is one or more of: injuryto articular cartilage; osteoarthritis; costochondritis; herniation;achondroplasia; relapsing polychondritis; benign or non-cancerouschondroma; and, malignant or cancerous chondrosarcoma.

Further provided herein is a method for inducing chondrogenesis leadingto cartilage formation or chondrogenesis leading to cartilage formationthat further mediates formation of new bone tissue in a vertebrate, themethod comprising administering a therapeutically effective amount of apharmaceutical composition as described herein to the vertebrate.

In certain embodiments, the administration is local or systemic.

Further provided herein is a method for promoting chondrogenesis at asite of skeletal surgery in a vertebrate, the method comprisingdelivering a pharmaceutical composition as described herein at the siteof skeletal surgery wherein such delivery induces chondrogenesis leadingto cartilage formation at the site or chondrogenesis leading tocartilage formation that further mediates formation of new bone tissueat the site.

In accordance with the present disclosure, there are provided hereincompounds that are analogs of PPARδ and 20-OH-PGE₂. The analog compoundsserve as either agonists to compounds that promote bone formation orantagonists to compounds that induce adipogenesis.

The first group of compounds provided herein, PPARδ affinity ligandanalogs, have the structural formula of Formula I:

wherein X is S, O, or NH; R is OCH₂R³, CH═CHR³, or (CH₂)_(n)R³, where nis 0, 1, 2, or 3 and R³ is carboxylic acid, sulfonic acid, an acidicsulfonamide, or pharmacophoric mimics thereof; R¹ is H, CH₃, or CF₃; andR² is H, alkyl, substituted alkyl, or halide; provided that when R isOCH₂CO₂H, (i) either R¹ is not para-CF₃ or R² is not H, and (ii) eitherR¹ is not meta-CH₃ or R² is not para-tert-butyl. Also provided aresalts, isomers, stereoisomers, enantiomers, racemates, solvates,hydrates, polymorphs, and prodrugs of Formula I.

PPARγ is a nuclear receptor protein that functions as a transcriptionfactor regulating the expression of genes. PPARγ plays a vital role inthe regulation of cellular differentiation and metabolism. For example,PPARγ activates fat metabolism to prevent obesity. The analogsencompassed by Formula I, some of which serve as PPARγ agonists, alsoinhibit adipogenesis and stimulate fat metabolism to prevent obesity.Provided herein are methods of increasing bone density involvingadministering the PPARγ agonist compounds described.

In certain instances, the compounds of Formula I are also, oralternatively, PPARγ agonists. In certain other instances, the compoundsof Formula I are both PPARγ agonists and PPARγ agonists. The compoundsof Formula I have a structure-activity relationship (SAR) thatdemonstrates the importance of the substitution pattern on the phenylring having the R group. The addition of a trifluoromethyl group toeither the para- or meta-position of this phenyl ring endows selectivityfor PPARγ over PPARγ, as compared to the molecule having no substitutionin this location. (FIG. 15.) This is distinguishable from the data inthe literature, and indicates that the meta-CF₃ group is a uniquelypreferred embodiment within this specific SAR series.

Scheme 1 and Scheme 2, shown in FIG. 12 and FIG. 13, respectively,illustrate non-limiting examples of synthetic routes to produce PPARγagonists. Within this group of compounds are described compounds 6a, 6b,and 6c. These analogs have the structural formula shown below as FormulaI-A.

Compound 6a has the structural formula of Formula I-A, where R₁ and R₂are each hydrogen. Compound 6b has the structural formula of FormulaI-A, where R₁ is CF₃ and R₂ is hydrogen. Compound 6c has the structuralformula of Formula I-A, where R₁ is hydrogen and R₂ is CF₃.

By way of a non-limiting example, compounds 6a-6c can be prepared viathe synthetic route shown in Scheme 1, FIG. 12. To begin, a carboxylicacid ethyl ester 1 is prepared by adding a chloroacetoacetate to athiobenzamide. Then, LiAlH₄ in THF is added to the carboxylic acid ethylester 1 to yield a thiazole alcohol 2. Triethylamine is added to thethiazolemethanol 2 in anhydrous dichloromethane, and to the resultingmixture is added methanesulfonyl chloride. The purified product is athiazole 4. A hydroxycinnamic acid methyl ester 3 is prepared by addingsulfuric acid to p-coumaric acid in anhydrous methanol. To a solution ofthe ester 3 and the thiazole 4 is added cesium carbonate. The product isa methyl ester 5. NaOH is added to the methyl ester 5 to yield compound6, wherein R₁ is H, CF₃, or CH₃, and R₂ is H, CF₃, alkyl, or substitutedalkyl.

The second group of compounds provided herein, 20-OH-PGE₂ analogs, havethe structural formula of Formula II:

wherein R is carboxylic acid, sulfonic acid, an acidic sulfonamide, orpharmacophoric mimics thereof; R¹ is H, CH₃, OH, CH₂OH; and n is 1, 2,or 3; provided that when R is CO₂H, either R¹ is not CH₂OH or n is not3. Further provided are salts, isomers, stereoisomers, enantiomers,racemates, solvates, hydrates, polymorphs, and prodrugs of Formula II.Compounds of Formula II are 20-OH-PGE₂ antagonists.

The significance of 20-OH-PGE₂ antagonists begins with20-hydroxy-5,8,11,14-eicosatetraeonic acid (20-HETE), which issynthesized by P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid.20-HETE is a primary eicosanoid in the microcirculation that plays arole in the regulation of vascular tone and renal tubular homeostasis.20-HETE has been shown to stimulate the production of superoxides andinflammatory cytokines, as well as inhibit endothelial eNOS and increaseoxidative stress. 20-HETE thus plays a role in the regulation of adiposetissue.

20-HETE induces oxidative stress and is associated with increased bodymass index (BMI) and the metabolic syndrome. 20-HETE has also been shownto mediate cellular proliferation, angiogenesis, and inflammation, allof which may materially contribute to the process of adipogenesis.20-HETE is metabolized by the cyclooxygenase (COX) pathway—therate-limiting enzyme that catalyzes the conversion of arachidonic acidinto prostaglandins—into 20-OH-endoperoxides and consequently into20-OH-PGE₂. 20-OH-PGE₂, as shown by the examples described below, is apotent inducer of adipogocity. The compounds of Formula II, which areantagonists of 20-OH-PGE₂, thus serve to prevent the inducement ofadipogenesis and thereby stimulate bone formation through osteogenesisor cartilage formation through chondrogenesis.

Scheme 3, shown in FIG. 14, illustrates a shortened synthetic route forproducing 20-OH-PGE₂ antagonists. Alternatively, a longer, 17-stepsynthesis of these compounds is possible.

It is intended that any of the compounds disclosed herein could be usedin a medication, a food additive, an injection, or a surgical implantdesigned to treat, ameliorate, or modify, osteoporosis, osteoarthritis,metabolic bone disease, and/or fracture management problems. Thecompounds of the present disclosure could be used to enhance the naturalpathways to direct a patient's own mesenchymal stem cells toward boneand cartilage formation over adipose formation, thereby preventingand/or treating these underlying conditions. The compounds could also beincorporated into a pharmaceutical composition, or could be used totreat isolated stem cells that are then administered to a patient inneed thereof.

Pharmaceutical Compositions

Pharmaceutical compositions of the present disclosure comprise aneffective amount of a compound disclosed herein, and/or additionalagents, dissolved or dispersed in a pharmaceutically acceptable carrier.The phrases “pharmaceutical” or “pharmacologically acceptable” refers tomolecular entities and compositions that produce no adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human. The preparation of a pharmaceutical composition thatcontains at least one compound or additional active ingredient will beknown to those of skill in the art in light of the present disclosure,as exemplified by Remington's Pharmaceutical Sciences, 2003,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it is understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

A composition disclosed herein may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. Compositions disclosed herein can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, intraosseously, periprosthetically, topically,intramuscularly, subcutaneously, mucosally, intraosseosly,periprosthetically, in utero, orally, topically, locally, via inhalation(e.g., aerosol inhalation), by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 2003, incorporated herein byreference).

The actual dosage amount of a composition disclosed herein administeredto an animal or human patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a composition herein and/or additional agent isformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsules, theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In further embodiments, a composition described herein may beadministered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered, for example but not limited to, intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally (U.S. Pat. Nos. 6,753,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363 are each specificallyincorporated herein by reference in their entirety).

Solutions of the compositions disclosed herein as free bases orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations may contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In most cases, the form must be sterileand must be fluid to the extent that easy injectability exists. Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersion,and/or by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, such as, but not limited to, parabens, chlorobutanol,phenol, sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption such as, for example, aluminum monostearate or gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating thecompositions in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized compositions into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, some methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. A powderedcomposition is combined with a liquid carrier such as, e.g., water or asaline solution, with or without a stabilizing agent.

In other embodiments, the compositions may be formulated foradministration via various miscellaneous routes, for example, topical(i.e., transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or via inhalation.

Pharmaceutical compositions for topical administration may include thecompositions formulated for a medicated application such as an ointment,paste, cream, or powder. Ointments include all oleaginous, adsorption,emulsion, and water-soluble based compositions for topical application,while creams and lotions are those compositions that include an emulsionbase only. Topically administered medications may contain a penetrationenhancer to facilitate adsorption of the active ingredients through theskin. Suitable penetration enhancers include glycerin, alcohols, alkylmethyl sulfoxides, pyrrolidones and laurocapram. Possible bases forcompositions for topical application include polyethylene glycol,lanolin, cold cream and petrolatum as well as any other suitableabsorption, emulsion or water-soluble ointment base. Topicalpreparations may also include emulsifiers, gelling agents, andantimicrobial preservatives as necessary to preserve the composition andprovide for a homogenous mixture. Transdermal administration of thecompositions may also comprise the use of a “patch.” For example, thepatch may supply one or more compositions at a predetermined rate and ina continuous manner over a fixed period of time.

In certain embodiments, the compositions may be delivered by eye drops,intranasal sprays, inhalation, and/or other aerosol delivery vehicles.Methods for delivering compositions directly to the lungs via nasalaerosol sprays has been described in U.S. Pat. Nos. 5,756,353 and5,804,212 (each specifically incorporated herein by reference in theirentirety). Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts and could be employed to deliverthe compositions described herein. Likewise, transmucosal drug deliveryin the form of a polytetrafluoroetheylene support matrix is described inU.S. Pat. No. 5,780,045 (specifically incorporated herein by referencein its entirety), and could be employed to deliver the compositionsdescribed herein.

It is further envisioned the compositions disclosed herein may bedelivered via an aerosol. The term aerosol refers to a colloidal systemof finely divided solid or liquid particles dispersed in a liquefied orpressurized gas propellant. The typical aerosol for inhalation consistsof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

Kits

It is further intended the compounds disclosed herein could be packagedin the form of a kit containing a single or separate containers. Manyembodiments of such kits are possible. For instance, a kit could housetwo containers, the first container comprising a compound of Formula I,and the second container comprising a compound of Formula II. By way offurther non-limiting example, a kit could have a first container housinga solution comprising one or more compounds of Formula I and Formula II,and a second container comprising a syringe configured to inject thesolution. As another example, a kit for the preparation of apharmaceutical composition could have a first container housing one ormore compounds of Formula I and Formula II, and a second containerhousing a pharmaceutically acceptable carrier, excipient, diluent, oradjuvant. Many other variations and embodiments of such kits areenvisioned. The kits typically further include instructions for usingthe components of the kit to practice the subject methods. Theinstructions for practicing the subject methods are generally recordedon a suitable recording medium. For example, the instructions may bepresent in the kits as a package insert or in the labeling of thecontainer of the kit or components thereof. In other embodiments, theinstructions are present as an electronic storage data file present on asuitable computer readable storage medium, such as a flash drive,CD-ROM, or diskette. In other embodiments, the actual instructions arenot present in the kit, but means for obtaining the instructions from aremote source, such as via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

EXAMPLES Example 1—Differential Effects of 20-OH-PGE₂ and 20-HETE onAdipogenesis

Frozen bone marrow mononuclear cells were purchased from Allcells(Emeryville, Calif.). After thawing, mononuclear cells were resuspendedin an α-minimal essential medium (α-MEM, Invitrogen, Carlsbad, Calif.)supplemented with 10% heat inactivated fetal bovine serum (FBS,Invitrogen) and 1% antibiotics and antimycotic (Invitrogen). The cellswere plated at a density of 1-5×10⁶ cells per 100 cm² dish. The cultureswere maintained at 37° C. in a 5% CO₂ incubator. The medium was changedafter 48 hours and every 3-4 days thereafter. When the mesenchymal stemcells (MSCs) were confluent, the cells were recovered by the addition of0.25% trypsin/EDTA (Invitrogen). MSCs were plated in either a 75 cm²flask or a 24-well plate and cultured in a α-MEM with 20% FBS up to adensity of 2.0×10⁴ cells/cm². The medium was replaced with adipogenicmedium, and the cells were cultured for an additional 14 days. Theadipogenic media consisted of complete culture medium supplemented withDMEM-high glucose, 10% (v/v) FBS, 10 μg/mL insulin, 0.5 mM dexamethasone(Sigma-Aldrich, St. Louis, Mo.), and 1% antibiotics and antimycotic(Invitrogen) in the presence and absence of the COX-1 inhibitor(2-valeryloxybenzoic acid, Cayman, Ann Arbor, Mich.) and the COX-2inhibitor(3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazol, Cayman)with and without 20-HETE, 20-HETE agonist (20-5,14-HEDE), or 20-OH-PGE₂.20-HETE, 20-HETE agonist, and 20-OH-PGE₂ were added three times a weekat concentrations of 0.1 μM and 1 μM. Inhibitors of COX-1 and COX-2 wereadded three times a week at a dose of 100 μM and 5 μM, respectively.

At day 14 of adipogenesis, 0.21% Oil Red O in 100% isopropanol(Sigma-Aldrich) was used. Briefly, adipocytes were fixed in 10%formaldehyde, washed in Oil Red O for 10 minutes, and rinsed with 60%isopropanol (Sigma-Aldrich). The Oil Red O was then eluted by adding100% isopropanol for 10 minutes, and OD was measured at 490 nm for 0.5 sreading. MSC-derived adipocytes were measured by Oil Red O staining(OD=490 nm) after day 14. Each value of Oil Red O staining wasnormalized by cell numbers (values at OD=490 nm).

Lipid droplets were measured using an ImagePro Analyzer (MediaCybernetics Corporation, Silver Springs, Md.). The MSC-derivedadipocytes were treated with increasing concentration of 20-OH-PGE₂(1-1,000 nM) every alternate day for 14 days. To quantify the number andsize of the lipid droplets in these images, a proprietary algorithm wasdeveloped that segments circular staining patterns. The algorithm wasthen applied to images obtained from the lipid optical channel for cellsexposed to different doses of 20-OH-PGE₂.

Total RNA was isolated using the RNeasy mini kit (Qiagen, Valencia,Calif.) according to the manufacturer's instructions. First-strand cDNAwas synthesized with Roche reverse transcription reagents. Total RNA(0.5-1 μg) was analyzed by real-time PCR. The quantitative real-timepolymerase chain reaction (qRT-PCR) was performed with the TaqMan geneexpression assay on an Applied Biosystems 7500 fast real-time PCR systemaccording to the manufacturer's recommended protocol (AppliedBiosystems, Foster City, Calif.). Each reaction was run in triplicate.The comparative threshold cycle (CT) method was used to calculate theamplification fold as specified by the manufacturer.

Western blot analysis of adipocyte cell lysate was carried out. Cellswere placed in a homogenization buffer, and homogenates were centrifugedat 27,000 g for 10 minutes at 4° C. The supernatant was used for themeasurements of COX-1, COX-2, PPARγ, Mest, and β-catenin protein levels.The levels were quantified by scanning densitometry using an imagingdensitometer, normalized to the levels of total protein.

PGE₂ levels were determined in the culture supernatant. Multiple assayswere conducted for quantification of the proteins (AssayGate Inc.,Ijamsville, Md.). All measurements were performed in triplicate.

Statistical significance between experimental groups was determined bythe Fisher method of analysis of multiple comparisons (P<0.05 wasregarded as significant). For comparison between treatment groups, thenull hypothesis was tested by either a single-factor ANOVA for multiplegroups or the unpaired t-test for two groups. Data are presented asmean±SEM. Differences between experimental groups were evaluated withANOVA with Bonferroni corrections. Statistical significance was regardedas significant at P<0.05.

The expression levels of CYP4-ω-hydroxylases were determined in MSCbefore and before completion of adipogensis as shown in FIGS. 1A-1B. MSCexpressed relatively high mRNA levels of CYP4A11 and CYP4F2 (the othermajor 20-HETE producing CYP-4-ω-hydroxylases in humans) before the startof adipogenic differentiation. In adipocytes derived from MSC, mRNAlevels of these hydroxylases were nearly undetectable. To evaluate COXactivity in MSC exposed to adipogenic environment, PGE₂ levels weredetermined in conditioned media (FIG. 1C). COX-1 and COX-2 inhibitorsdecreased PGE₂ levels compared with levels in the conditioned mediawithout indomethacin. Addition of indomethacin, which is a dual COX-1and COX-2 inhibitor, further decreased PGE₂ levels, as shown in FIG. 1C.

The effect of 20-HETE on lipid accumulation in MSC-derived adipocyteswas examined in the presence and the absence of either a COX-1 inhibitoror a COX-2 inhibitor. As seen in FIGS. 2A-2B, 20-HETE enhanced lipidaccumulation in cells exposed to a COX-1 inhibitor but not in cellsexposed to COX-2 inhibitor. The absence of such an effect on 20-HETE incells treated with a COX-2 inhibitor indicates that a COX-2-derived20-HETE metabolic product plays a role in mediating these enhancedlipogenic effects. The direct effect of 20-HETE on lipid accumulation inMSCs derived adipocytes was further refuted by the inability of a20-HETE agonist [sodium 2-((5Z,14Z—)-20-hydroxyicosa-5,14-dienamido)acetate, 20-HEDE] to mimic the effects of exogenous 20-HETE, as shown inFIG. 3. Results show that in the presence of COX-1 and COX-2 inhibitors,the 20-HETE agonist 20-HEDE had no significant effect on adipogenesis,thus further indicating that a COX-2-derived metabolic product has anenhanced adipogenic effect. Subsequently, addition of the microsomalPGE₂ synthase inhibitor CAY10526 abolished the adipogenic effect of20-HETE in the presence of a COX-1 inhibitor, as shown in FIGS. 4A-4B.

The effects of 20-OH-PGE₂ on adipogensis in the presence or absence ofCOX-1 and COX-2 inhibitors were evaluated. 20-OH-PGE₂ stimulated(P>0.05) adipogenesis 4-fold as measured by lipid accumulation (FIGS.5A-5B). Neither COX-1 inhibitor nor COX-2 inhibitor, alone or together,prevented 20-OH-PGE₂-mediated increase in adipogenesis, indicating aCOX-independent action of this metabolite. As seen from FIG. 6A, theeffect of 20-OH-PGE₂ was concentration dependent. 20-OH-PGE₂significantly increased lipid accumulation at 50 nM and had maximaleffect at 500 nM. In addition, 20-OH-PGE₂ stimulated adipocytehypertrophy as measured by lipid droplet size. As seen in FIG. 6B, lipiddroplet size increased 2- and 3-fold in response to concentrations of100 nM and 1,000 nM, respectively, of 20-OH-PGE₂. These results indicatethat the COX-2 metabolite 20-OH-PGE₂ plays a significant role ininducing adipogenesis and that its effect may be antagonized by PGE₂.

The effects of 20-OH-PGE₂ on β-catenin, PPARγ, and Mest expression asadipogenic differentiation markers, in the presence and absence of COX-1and COX-2 inhibitors, were examined. (FIGS. 7A-7D.) Densitometryanalysis showed that the expression of PPARγ and Mest (FIG. 7B and FIG.7C, respectively) was significantly increased in the presence of20-OH-PGE₂ when the cells were treated with COX-1 inhibitor, COX-2inhibitor, or both. In contrast, as seen in FIG. 7D, β-cateninexpression significantly decreased in the presence of 20-OH-PGE₂ whenthe cells were treated with COX-1 inhibitor, COX-2 inhibitor, or both.Similar data were obtained with 20-HETE, except that in the presence ofa COX-2 inhibitor, the effect was blunted.

The effect of 20-OH-PGE₂ on mesenchymal stem cells was evaluated. Asshown in FIGS. 8-11, 20-OH-PGE₂ blocked bone formation, meaningmesenchymal stem cells form fat over bone upon contact with 20-OH-PGE₂.FIG. 8 is a summary of the effects of 20-OH-PGE₂ with increasingconcentration on both adipogenesis and osteogenesis. As seen from thestained mesenchymal cell pictures in this figure, fat formationincreased with increasing 20-OH-PGE₂ concentration, and bone formationdecreased with increasing 20-OH-PGE₂ concentration.

FIG. 9 shows images of stained mesenchymal cells with varyingconcentrations of 20-OH-PGE₂ and a PPARγ agonist. The images revealadipogenesis increased with increasing concentrations of 20-OH-PGE₂, butdecreased with increasing concentrations of the PPARγ agonist. Theimages further reveal an increase in osteogenesis with increasingconcentration of the PPARγ agonist.

FIG. 10A depicts the osteogenesis effects of 20-OH-PGE₂ on mesenchymalcells stained with Alizarin S and Oil Red O. FIG. 10B shows the effectsof 20-OH-PGE₂ on osteogenesis from BM-derived mesenchymal stem cells. Asseen from the chart in this figure, osteogenesis decreased withincreasing concentration of 20-OH-PGE₂. This also shows thatosteogenesis decreased with increasing concentration of 20-OH-PGE₂.

Example 2—Effects of GW0742 and GSK0660

hBM-derived MSCs were treated with GW0742 (a PPARγ agonist) and GSK0660(a PPARγ antagonist) every 2 days during osteogenesis. Cells werestained with 2% Alizarin S, and images were taken from 24 well platesunder a microscope. FIG. 11A shows four of these microscope images.Mineralization was quantified with Metamorph software. As seen in FIG.11B, increased red staining on the PPARγ agonist (GW0742) was observedat 0.1 μM and 1 μM, indicating an increase in osteogenesis, anddecreased red staining on the PPARγ antagonist (GSK0660) was observed at1 μM, indicating a decrease in osteogenesis.

Example 3—Synthesis of Target Compound 6a

Compound 6a was prepared via Scheme 1, shown in FIG. 12.

Synthesis of 4-methyl-2-phenylthiazole-5-carboxylic acid ethyl ester(1a)

To a suspension of thiobenzamide (6.05 g, 0.044 mol) in 95% ethanol wasadded ethyl 2-chloroacetoacetate (6.10 mL, 0.044 mol), and the mixturewas stirred at reflux temperature for 26 hours. The reaction mixture wasconcentrated under reduced pressure and the resulting residue wassuspended in ice-cold hexane and stirred for 20 minutes. The suspensionwas filtered and collected as a cream-colored solid (7.434 g, 0.030 mol,68.3%). TLC R_(f) (25% EtOAc/Hexane)=0.63. Mp 84-87° C. ¹H NMR (CDCl₃,600 MHz): δ ppm 8.19 (2H, d, J=7.32 Hz), 7.56 (1H, t, J=7.32), 7.53 (2H,t, J=7.08), 4.41 (2H, q, J=7.14), 2.94 (3H, s), 1.41 (3H, t, J=7.14).¹³C NMR (CDCl₃, 150 MHz): δ (ppm) 171.25, 161.07, 157.83, 133.21,129.82, 128.10, 122.55, 62.37, 16.29, 14.52 ppm.

Synthesis of 4-methyl-2-phenyl-5-thiazolemethanol (2a)

To a stirred solution of ethyl ester 1a (0.303 g, 1.237 mmol) inanhydrous THF (1 mL) at 0° C. was added 2M lithium aluminum hydridesolution in THF (1.24 ml, 2.48 mmol). The resulting mixture was stirredunder argon at 0° C. for 1.5 hours. The reaction mixture was quenched bythe careful addition of 0.5 ml of water, followed by 2.5 ml of ethylacetate and 0.92 g of anhydrous sodium sulfate. The mixture was stirredfor 15 minutes and was filtered and concentrated under reduced pressureto give 2a as a light-yellow solid (0.215 g, 1.053 mmol, 85.1%). TLCR_(f) (25% EtOAc/Hexane)=0.11. Mp 101-102° C. ¹H NMR (CDCl₃, 600 MHz): δppm 7.88 (2H, d, J=7.92 Hz), 7.41 (3H, m), 4.79 (2H, s), 2.94 (1H, s),2.41 (3H, s).

Synthesis of 4-hydroxycinnamic acid methyl ester (3)

To a stirred solution of p-coumaric acid (0.704 g, 4.288 mmol) inanhydrous methanol (10 ml) was added concentrated H₂SO₄ (1 ml) and washeated at reflux temperature for 20 hours. The mixture was cooled toroom temperature and concentrated under reduced pressure. The residuewas dissolved in ethyl acetate, washed with water, dried with Na₂SO₄,and concentrated. The residue was purified by column chromatography onsilica gel to give 3 as a white solid (0.574 g, 3.221 mmol, 75.1%). TLCR_(f) (25% EtOAc/Hexane)=0.18. Mp 137-138° C. ¹H NMR (CDCl₃, 600 MHz): δppm 7.65 (1H, d, J=16.02 Hz), 7.44 (2H, d, J=8.58), 6.86 (2H, d,J=8.58), 6.31 (1H, d, J=15.98), 5.57 (1H, s), 3.81 (3H, s).

Synthesis of 5-(chloromethyl)-4-methyl-2-phenyl-thiazole (4a)

To a stirred solution of alcohol 2a (4.095 g, 0.019 mol) in anhydrousdichloromethane (100 ml) was added triethylamine (5.50 ml, 0.039 mol).The resulting mixture was cooled to 4° C. and methanesulfonyl chloride(2.30 ml, 0.029 mol) was slowly added. The mixture was stirred at 4° C.for 24 hours and then diluted with 100 ml dichloromethane, washed withsaturated NaHCO₃ solution, water, brine, dried with Na₂SO₄, andconcentrated. The residue was purified by column chromatography onsilica gel with 10% ethyl acetate/hexane to give 4a as a light yellowsolid (2.850 g, 0.013 mol, 64.0%). TLC R_(f) (25% EtOAc/Hexane)=0.57. Mp89-90° C. ¹H NMR (CDCl₃, 600 MHz): δ ppm 7.90 (2H, m), 7.43 (3H, m),4.80 (2H, s), 2.50 (3H, s).

Synthesis of [4-[[4-methyl-2-phenylthiazol-5-yl]methyl]methoxy]cinnamicacid methyl ester (5a)

To a stirred solution of methyl ester 3 (0.142 g, 0.797 mmol) andchloromethyl 4a (0.150 g, 0.670 mmol) in anhydrous acetonitrile (5 ml)was added cesium carbonate with partial solubility. The resultingmixture was stirred for 24 hours at room temperature at which TLC showedthat the chloromethyl 4a had been consumed. The reaction mixture wasconcentrated and the residue was dissolved in ethyl acetate and washedwith water, brine, dried with Na₂SO₄, and concentrated. Columnchromatography on silica gel failed to give a pure product and the crudewhite solid 5 collected was moved to the next step without furtherpurification.

Synthesis of [4-[[4-methyl-2-phenylthiazol-5-yl]methyl]methoxy]cinnamicacid (6a)

To a stirred solution of methyl ester 5 was added dropwise 3M NaOH.After 20 hours, the mixture was acidified with 1M HCl to a pH=1-2 andconcentrated. The residue was suspended in ethyl acetate and washed withwater and brine. The aqueous phase was extracted with a separate portionof ethyl acetate and the organic phases were combined, dried withNa₂SO₄, and concentrated. The residue was purified by columnchromatography on silica gel to give 6a as a white solid (0.061 g, 0.173mmol, 41.7%). TLC R_(f) (50% EtOAc/Hexane)=0.17. Mp 209-211° C. ¹H NMR(Acetone-d6, 600 MHz): δ ppm 7.91 (2H, m), 7.67 (2H, d, J=8.76), 7.55(1H, d, 15.96), 7.49 (3H, m), 7.09 (2H, d, J=8.82), 6.41 (1H, d,J=15.96), 5.38 (2H, s), 2.46 (4H, s).

Example 4—Synthesis of Target Compound 6b

Compound 6b was prepared via Scheme 1, shown in FIG. 12.

Synthesis of4-methyl-2-[4-(trifluoromethyl)phenyl]-thiazole-5-carboxylic acid ethylester (1b)

In analogy to the procedure described above to produce 1a,4-(trifluoromethyl)thiobenzamide (1.065 g, 5.190 mmol) was treated withethyl-2-chloroacetoacetate in 95% ethanol to give 1b as a cream-coloredsolid (1.148 g, 3.644 mmol, 70.2%). TLC R_(f) (25% EtOAc/Hexane)=0.69.Mp 89-89.5° C.

Synthesis of 4-methyl-2-[4-(trifluoromethyl)phenyl]-thiazole-5-methanol(2b)

In analogy to the procedure described above to produce 2a, ethyl ester1b (1.320 g, 4.190 mmol) was treated with 2M LiAlH₄ solution in THF togive 2b as a yellow solid (0.904 g, 3.308 mmol, 79.0%). TLC R_(f) (25%EtOAc/Hexane)=0.16. Mp 121.5-122° C.

Synthesis of5-(chloromethyl)-4-methyl-2-[4-(trifluoromethyl)phenyl]-thiazole (4b)

In analogy to the procedure described above to produce 4a, alcohol 2b(0.883 g, 3.231 mmol) was treated with methanesulfonyl chloride andtriethylamine in dry DCM to give 4b as a light yellow solid (0.790 g,2.708 mmol, 83.8%). TLC R_(f) (25% EtOAc/Hexane)=0.53. Mp 68.5-69° C.

Synthesis of[4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-thiazol-5-yl]methyl]methoxy]cinnamicacid methyl ester (5b)

In analogy to the procedure described above to produce 5a, chloromethyl4b (0.331 g, 1.135 mmol) and methyl ester 3 were treated with cesiumcarbonate in anhydrous acetonitrile to give 5b as a light yellow solid(0.365 g, 0.842 mmol, 74.3%). TLC R_(f) (25% EtOAc/Hexane)=0.32. Mp153-155° C.

Synthesis of[4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-thiazol-5-yl]methyl]methoxy]cinnamicacid (6b)

In analogy to the procedure described above to produce 6a, methyl ester5b (0.202 g, 0.466 mmol) was treated with 3M NaOH in 95% ethanol to give6b as a white solid (0.048 g, 0.114 mmol, 24.6%). TLC R_(f) (50%EtOAc/Hexane)=0.15. Mp 224-225° C. ¹H NMR (Acetone-d6, 600 MHz): δ ppm8.18 (2H, d, J=8.10), 7.84 (2H, d, J=8.22), 7.68 (2H, d, J=8.76), 7.64(1H, d, J=16.02), 7.13 (2H, d, J=8.82), 6.42 (1H, d, J=15.96), 5.44 (2H,s), 2.52 (3H, s).

Example 5—Synthesis of Target Compound 6c

Compound 6c was prepared via Scheme 1, shown in FIG. 12.

Synthesis of4-methyl-2-[3-(trifluoromethyl)phenyl]-thiazole-5-carboxylic acid ethylester (1c)

In analogy to the procedure described above to produce 1a,3-(trifluoromethyl)thiobenzamide (0.501 g, 2.442 mmol) was treated withethyl-2-chloroacetoacetate in 95% ethanol to give 1c as a cream-coloredsolid (0.567 g, 1.790 mmol, 73.7%). TLC R_(f) (25% EtOAc/Hexane)=0.63.Mp 90-91° C.

Synthesis of 4-methyl-2-[3-(trifluoromethyl)phenyl]-thiazole-5-methanol(2c)

In analogy to the procedure described above to produce 2a, ethyl ester1c (2.053 g, 6.516 mmol) was treated with 2M LiAlH₄ solution in THF togive 2c as a yellow oil (1.273 g, 4.658 mmol, 71.5%). TLC R_(f) (25%EtOAc/Hexane)=0.16.

Synthesis of5-(chloromethyl)-4-methyl-2-[3-(trifluoromethyl)phenyl]-thiazole (4c)

In analogy to the procedure described above to produce 4a, alcohol 2c(1.295 g, 4.739 mmol) was treated with methanesulfonyl chloride andtriethylamine in dry DCM to give 4c as a light yellow solid (0.830 g,2.846 mmol, 60.1%). TLC R_(f) (25% EtOAc/Hexane)=0.61. Mp 42-43° C.

Synthesis of[4-[[4-methyl-2-[3-(trifluoromethyl)phenyl]-thiazol-5-yl]methyl]methoxy]cinnamicacid methyl ester (5c)

In analogy to the procedure described above to produce 5a, chloromethyl4c (0.472 g, 1.618 mmol) and methyl ester 3 were treated with cesiumcarbonate in anhydrous acetonitrile to give 5c as a yellow-white solid(0.551 g, 1.270 mmol, 80.5%). TLC R_(f) (25% EtOAc/Hexane)=0.30. Mp125-127° C.

Synthesis of[4-[[4-methyl-2-[3-(trifluoromethyl)phenyl]-thiazol-5-yl]methyl]methoxy]cinnamicacid (6c)

In analogy to the procedure described above to produce 6a, methyl ester5c (0.207 g, 0.477 mmol) was treated with 3M NaOH in 95% ethanol to give6c as a white solid (0.138 g, 0.329 mmol, 69.0%). TLC R_(f) (50%EtOAc/Hexane)=0.40. Mp 179.5-181° C. ¹H NMR (Acetone-d6, 600 MHz): δ ppm8.29 (1H, s), 8.23 (1H, d, J=7.8), 7.84 (1H, d, J=7.8), 7.76 (1H, t,J=7.8), 7.69 (2H, d, J=8.7), 7.67 (1H, d, J=16.02), 7.15 (2H, d,J=8.76), 6.44 (1H, d, J=15.96), 5.46 (2H, s), 2.54 (3H, s).

Example 6—Effects of Compounds 6a-6c on Adipogenesis and Osteogenesis

The effects of compounds 6a, 6b, and 6c on adipogenesis and osteogenesiswere evaluated. MSCs were treated with compounds 6a, 6b, and 6c asdescribed above for GW0742. FIG. 15 shows a table summarizing theresults of these assays. As seen in FIG. 15, compound 6a increasedadipogenesis versus osteogenesis, and compound 6c increased osteogenesisversus adipogenesis. These results are also shown in FIG. 16, alongsidethe effects of GW0742 described above.

Certain embodiments of the compounds, compositions, and methodsdisclosed herein are defined in the above examples. It should beunderstood that these examples, while indicating particular embodimentsof the invention, are given by way of illustration only. From the abovediscussion and these examples, one skilled in the art can ascertain theessential characteristics of this disclosure, and without departing fromthe spirit and scope thereof, can make various changes and modificationsto adapt the compositions and methods described herein to various usagesand conditions. Various changes may be made and equivalents may besubstituted for elements thereof without departing from the essentialscope of the disclosure. In addition, many modifications may be made toadapt a particular situation or material to the teachings of thedisclosure without departing from the essential scope thereof.

What is claimed is:
 1. A method of treating osteoporosis by inducingosteogenesis in a subject having osteoporosis, the method comprising:administering an effective amount of a pharmaceutical composition to ahuman patient in need thereof sufficient to induce osteogenesis; thepharmaceutical composition comprising a peroxisome proliferatoractivated receptor (PPAR) compound in an amount sufficient to promptstem cells in the patient to contribute toward improving bone density,and a pharmaceutically acceptable carrier, excipient, diluent oradjuvant; wherein the PPAR compound has a chemical structure of FormulaI:

wherein: X is O; R is CH═CHR³, R³ is a carboxylic acid; R¹ is para-CF₃and R² is meta-H; and salts, isomers, solvates, hydrates, polymorphs andprodrugs thereof.
 2. The method of claim 1, wherein the administrationis by an intravenous, intramuscular or subcutaneous injection of liquidor gel formulations or delivery systems.
 3. The method of claim 1,wherein the administration is by the oral route.
 4. A method of treatingosteoporosis, the method comprising administering induced stem cells toa human patient in need thereof; the induced stem cells derived fromincubating stem cells with a pharmaceutical composition comprising aperoxisome proliferator activated receptor (PPAR) compound and apharmaceutically acceptable carrier, excipient, diluent or adjuvant;wherein the PPAR compound has a chemical structure of Formula I:

wherein X is O; R is CH═CHR³, R³ is a carboxylic acid; R¹ is para-CF₃and R² is meta-H; and salts, isomers, solvates, hydrates, polymorphs andprodrugs thereof.
 5. The method of claim 4, wherein the stem cells areeither harvested from the same patient or supplied from anothermammalian donor.
 6. The method of claim 5, wherein the administration isby an intravenous, intramuscular or subcutaneous injection of liquid orgel formulations or delivery systems.